目的建立丙型肝炎病毒(hepatitis C virus,HCV)抗体分片段检测方法。方法以葡聚糖T500为骨架,将亲和素和HRP标记于葡聚糖的不同位置上,再分别与含有生物素标签的HCV的核心抗原、NS3、NS4B、NS5反应(亲和素及HRP与HCV抗原的摩尔比均为1∶...目的建立丙型肝炎病毒(hepatitis C virus,HCV)抗体分片段检测方法。方法以葡聚糖T500为骨架,将亲和素和HRP标记于葡聚糖的不同位置上,再分别与含有生物素标签的HCV的核心抗原、NS3、NS4B、NS5反应(亲和素及HRP与HCV抗原的摩尔比均为1∶10),制备HCV抗原聚合物,同时结合免疫层析技术建立HCV抗体分片段检测方法。采用建立的方法检测100份正常人和100份HCV感染者的血清样本,分析层析膜上不同抗原条带组合出现的比例,计算特异度和灵敏度,根据特异度和灵敏度指标来确定阴阳性的判定标准。分别采用本实验建立的方法及商业化的蛋白芯片法检测196份临床血清样本,计算两种方法的符合率。结果 HCV抗体分片段检测方法检测200份临床标本的灵敏度为100. 0%,特异性为100. 0%,检测结果的判定标准为:2条带显色为阳性,1条带显色为可疑,需进一步确认。该方法与蛋白芯片法对196份临床血清样本的4种抗体片段检测结果符合率均达98. 0%以上。结论本研究成功建立了HCV抗体分片段检测方法,该方法具有良好的灵敏度及特异度,可用于HCV感染的诊断。展开更多
[Objective] This study aimed to establish a method for quantitative detection of mRNA transcriptional level of SS2 adhesive related-factors of Streptococcus suis serotype 2 （SS2） by fluorescent quantitative PCR. []V...[Objective] This study aimed to establish a method for quantitative detection of mRNA transcriptional level of SS2 adhesive related-factors of Streptococcus suis serotype 2 （SS2） by fluorescent quantitative PCR. []Vlethod] The gene fragments en- coding SS2 adhesive related-factors MRP, FBPS and CPS2J and a housekeeping gene aroA were amplified by reverse transcription PCR from the total RNA of SS2, cloned, and sequenced. The recombinant plasmids containing the target genes were constructed, and used as templates in Real-time PCR. [Result] Dynamic curves, stan- dard curves and melting curves of the adhesive related-factors and aroA were ob- tained by the optimized Real-time PCR system. The standard curves showed a good linear relationship between template copy number and circulation number, and the correlation coefficients （FF） of the standard curves were over 0.995. Also, these as- says were highly specific a^d there was single specific melting peak for every gene. Moreover, the assays were highly sensitive and had a detection limit of 1.0×102 copies in 1 μl of initial templates. Finally, it was highly repeatable and had a coeffi- cient of variation less than 2% for intra-assay. [Conclusion] This study will provide a way to reveal the adhesion mechanism of SS2 to different host cells at molecular level.展开更多
The main purpose of this study was to detect an association of cytoplasmic signal transducers and activators of transcription-1 （STAT1） with milk production traits in 472 Holstein and 283 Jersey cattle breeds of Tur...The main purpose of this study was to detect an association of cytoplasmic signal transducers and activators of transcription-1 （STAT1） with milk production traits in 472 Holstein and 283 Jersey cattle breeds of Turkey. This gene, located on chromosome 2, was chosen due to its role on development of mammary gland. A polymorphism of selected 314 bp allele fragment was detected by the restriction fragment length polymorphism analysis of polymerase chain reaction-amplified fragments （PCR-RFLP） method and also confirmed by DNA sequencing. The association tests were conducted between STAT1 genotypes and some economically important dairy traits. The genotypes for C/T as a single nucleotide polymorphism （SNP） were identified at interval 60 cM to 63 cM. The effects of STAT1 gene on milk production traits were not significant in Holstein cows, although animals with CT genotypes showed fairly close to significant value for the corrected 305 d milk yield. However, Jersey cows with/7＂ genotype were 2.07 kg higher for test-day milk yield （P 〈 0.05）, 0.13 kg for fat yield （P 〈 0.01） and 0.07 kg for protein yield （P 〈 0.05） compared with animals having CC and CT genotypes. Definitely, the further research should be conducted to search this gene intensively with larger samples to identify polymorphism and any association between the economically important traits and genotypic class in Holstein cows. Finally, based on the findings, it was concluded that STATI gene might be used as a potential candidate gene to improve milk yield and milk fat and protein contents in dairy cows breeding programs.展开更多
The AFLP(amplified fragment length polymorphism) technique was used to analyze and compare the genetic diversity of Tachypleus tridentatus from three south-eastern coastal sites of China(Pingtan,Hong Kong and Beihai)....The AFLP(amplified fragment length polymorphism) technique was used to analyze and compare the genetic diversity of Tachypleus tridentatus from three south-eastern coastal sites of China(Pingtan,Hong Kong and Beihai).Eight pairs of primers generated 361 loci,including 285 polymorphic loci.The ratio of polymorphic loci was 96.97%.Nei's genetic diversity index was 0.420 8 and the Shannon information index was 0.607 5,both of which were higher than that reported for many other arthropods.These results show that the genetic diversity detected was mainly caused by individual differences within a population.Genetic distance showed that the rational division of the three geographic populations of T.tridentatus along the south-eastern coast of China was not significant,in which the genetic distance was not proportional to the geographic distance.All three horseshoe crab populations may belong to a large group,and had a high degree of genetic similarity.The high level of genetic diversity obtained from the present AFLP analysis may be due to the large effective population size of the species in Chinese waters.展开更多
Based on rpoB gene micro array as target gene, we are going to use gene chip technology to test 24 mycobacterium standard specimens, 8 non-mycobacterium specimens and 86 mycobacterium clinical isolated specimens. As a...Based on rpoB gene micro array as target gene, we are going to use gene chip technology to test 24 mycobacterium standard specimens, 8 non-mycobacterium specimens and 86 mycobacterium clinical isolated specimens. As a result, after mycobacterium and non-mycobacterium standard specimens were duplicated by PCR, mycobacterium standard specimens reproduced 360bp DNA fragments; on the other hand, non-mycobacterium specimens did not reproduce any fragments except for hemolytic streptococcus and corynebacterium pseudodiphtheriticum which had the same results as mycobacterium standard specimens. Sensitive test is able to detect lpg tuberculosis mycobacterium DNA. The probe test showed that, among 21 oligonucleotide probes, probe-M. fortuitum and M. marinum were cross-hybrid; the other probes were specific. We used the new method to identify 126 mycobacterium clinical isolated specimens. The test results of this new method matched with conventional method. In conclusion, compared to the traditional method, the use of rpob gene chip technology to identify mycobacterium species will be faster, more accurate and higher value in application.展开更多
基金Supported by National Natural Science Foundation of China(31072155)Natural Science Foundation of Jiangsu Province(BK2010068)+1 种基金Fund for Independent Innovation of Agricultural Science in Jiangsu Province[CX(11)2060]Special Fund for Agroscientific Research in the Public Interest(201303041)~~
文摘[Objective] This study aimed to establish a method for quantitative detection of mRNA transcriptional level of SS2 adhesive related-factors of Streptococcus suis serotype 2 （SS2） by fluorescent quantitative PCR. []Vlethod] The gene fragments en- coding SS2 adhesive related-factors MRP, FBPS and CPS2J and a housekeeping gene aroA were amplified by reverse transcription PCR from the total RNA of SS2, cloned, and sequenced. The recombinant plasmids containing the target genes were constructed, and used as templates in Real-time PCR. [Result] Dynamic curves, stan- dard curves and melting curves of the adhesive related-factors and aroA were ob- tained by the optimized Real-time PCR system. The standard curves showed a good linear relationship between template copy number and circulation number, and the correlation coefficients （FF） of the standard curves were over 0.995. Also, these as- says were highly specific a^d there was single specific melting peak for every gene. Moreover, the assays were highly sensitive and had a detection limit of 1.0×102 copies in 1 μl of initial templates. Finally, it was highly repeatable and had a coeffi- cient of variation less than 2% for intra-assay. [Conclusion] This study will provide a way to reveal the adhesion mechanism of SS2 to different host cells at molecular level.
文摘The main purpose of this study was to detect an association of cytoplasmic signal transducers and activators of transcription-1 （STAT1） with milk production traits in 472 Holstein and 283 Jersey cattle breeds of Turkey. This gene, located on chromosome 2, was chosen due to its role on development of mammary gland. A polymorphism of selected 314 bp allele fragment was detected by the restriction fragment length polymorphism analysis of polymerase chain reaction-amplified fragments （PCR-RFLP） method and also confirmed by DNA sequencing. The association tests were conducted between STAT1 genotypes and some economically important dairy traits. The genotypes for C/T as a single nucleotide polymorphism （SNP） were identified at interval 60 cM to 63 cM. The effects of STAT1 gene on milk production traits were not significant in Holstein cows, although animals with CT genotypes showed fairly close to significant value for the corrected 305 d milk yield. However, Jersey cows with/7＂ genotype were 2.07 kg higher for test-day milk yield （P 〈 0.05）, 0.13 kg for fat yield （P 〈 0.01） and 0.07 kg for protein yield （P 〈 0.05） compared with animals having CC and CT genotypes. Definitely, the further research should be conducted to search this gene intensively with larger samples to identify polymorphism and any association between the economically important traits and genotypic class in Holstein cows. Finally, based on the findings, it was concluded that STATI gene might be used as a potential candidate gene to improve milk yield and milk fat and protein contents in dairy cows breeding programs.
基金Supported by the Conservation of Horseshoe Crabs in Hong Kong:Artificial Breeding and Public Awareness by Ocean Park Conservation Foundation Hong Kong (No. PJ 9230048)
文摘The AFLP(amplified fragment length polymorphism) technique was used to analyze and compare the genetic diversity of Tachypleus tridentatus from three south-eastern coastal sites of China(Pingtan,Hong Kong and Beihai).Eight pairs of primers generated 361 loci,including 285 polymorphic loci.The ratio of polymorphic loci was 96.97%.Nei's genetic diversity index was 0.420 8 and the Shannon information index was 0.607 5,both of which were higher than that reported for many other arthropods.These results show that the genetic diversity detected was mainly caused by individual differences within a population.Genetic distance showed that the rational division of the three geographic populations of T.tridentatus along the south-eastern coast of China was not significant,in which the genetic distance was not proportional to the geographic distance.All three horseshoe crab populations may belong to a large group,and had a high degree of genetic similarity.The high level of genetic diversity obtained from the present AFLP analysis may be due to the large effective population size of the species in Chinese waters.
文摘Based on rpoB gene micro array as target gene, we are going to use gene chip technology to test 24 mycobacterium standard specimens, 8 non-mycobacterium specimens and 86 mycobacterium clinical isolated specimens. As a result, after mycobacterium and non-mycobacterium standard specimens were duplicated by PCR, mycobacterium standard specimens reproduced 360bp DNA fragments; on the other hand, non-mycobacterium specimens did not reproduce any fragments except for hemolytic streptococcus and corynebacterium pseudodiphtheriticum which had the same results as mycobacterium standard specimens. Sensitive test is able to detect lpg tuberculosis mycobacterium DNA. The probe test showed that, among 21 oligonucleotide probes, probe-M. fortuitum and M. marinum were cross-hybrid; the other probes were specific. We used the new method to identify 126 mycobacterium clinical isolated specimens. The test results of this new method matched with conventional method. In conclusion, compared to the traditional method, the use of rpob gene chip technology to identify mycobacterium species will be faster, more accurate and higher value in application.
